Project Summary ? Project #3 The goal of this project is to characterize the influence on bone remodeling during adulthood and aging of extra-intestinal sites of expression of Tryptophan hydroxylase 1 (Tph1), the enzyme controlling the synthesis of two extracellular cues: serotonin and melatonin. Instead of the high bone mass/increased bone formation phenotype caused by Tph1 specific inactivation in gut cells we have observed that Tph1-/- mice show a mild increase in bone formation compensated by a mild increase in osteoclast number. This suggests that Tph1 expression outside the gut regulates the production of molecules that can counteract the effect on bone mass accrual caused by gut-derived serotonin. Although Tph1 is preferentially expressed in gut cells, its expression in the pineal gland is required for melatonin biosynthesis. Thus, a global inactivation of Tph1 causes an absence of both serotonin and melatonin. This led us to hypothesize that melatonin is a positive regulator of bone mass accrual that opposes the negative effect of gut- derived serotonin on bone formation. Consistent with this hypothesis, melatonin treatment of primary osteoblasts increases the expression of D-type cyclins, whose expression is instead down-regulated by gut- derived serotonin. Further analyses showed that melatonin could offset the effect of sympathetic signaling on CREB, ATF4 and their respective target genes in these cells. These preliminary studies suggest that melatonin could positively act on bone mass accrual by antagonizing the effect of both gut-derived serotonin and sympathetic signaling in osteoblasts, a contention that our project proposes to assess. A second observation we made was that Tph1+/- mice born from Tph1-/- mothers have a significantly lower bone mass at 3 months of age than Tph1+/- mice born from wild-type mothers. These data suggest that maternal serotonin and/or melatonin could be determinants of peak bone mass in the adult offspring. Serotonin does not cross the placenta but Tph1 expression in this tissue causes its release in fetal blood. Likewise, maternal melatonin reaches the fetal circulation. We therefore propose to evaluate whether maternally-produced melatonin and/or serotonin are required for proper bone mass acquisition and homeostasis in the offspring. Our specific aims are: 1) To assess through genetic means the role of melatonin produced by the pineal gland on bone biology. 2) To identify the receptor(s) and signaling pathway(s) mediating melatonin function(s) in osteoblasts in vivo. 3) To define the cellular and molecular bases of the influence exerted by maternal Tph1 expression on bone mass accrual in the offspring.